Epigenetics concerns the transmission of information from a cell or multicellular organism to its descendants without that information being encoded in the nucleotide sequence of genes. One mechanism by which epigenetic information is transmitted is via methylation of cytosine (C) bases in the genomic DNA of multicellular organisms.
DNA methylation in multicellular organisms occurs mainly at CpG dinucleotides, and has important regulatory functions in development and in the epigenetic control of gene expression, including genomic imprinting, X chromosome inactivation, the silencing of transposable elements, and possibly wider roles in silencing of genes in development. Loss of DNA methylation can occur during DNA replication by inactivation of the major maintenance methyltransferase Dnmt1. In addition, there are a number of examples in mammals and plants where demethylation occurs without replication of DNA, and hence is likely to be an active enzymatic process.
Active demethylation of DNA is thought to occur in a variety of biological systems in animals and plants, but the molecular mechanisms are not yet understood. In Arabidopsis, the DNA glycosylase Demeter has been shown to excise 5-methylcytosine from DNA and to be required for the activation of the maternal allele of Medea, an imprinted gene.
In cancer, it is well documented that the majority of tumour cells display abnormal DNA epigenetic imprints (Feinberg and Vogelstein, 1983). Studies have gone on to show that tumour suppressor genes within cancer cells are silenced by DNA methylation (Lyko and Brown, 2005). In gastric cancers, inactivation of the genes CDKN2A, CDH1, hMLH1 and RUNX3 has been described as resulting from DNA methylation in their respective promoters and it is believed that these and other genes may be methylated in response to infection by Helicobacter pylori (Ushijima, 2007).
Cancer is the second leading cause of death in the United States. An estimated 10.1 million Americans are living with a previous diagnosis of cancer. In 2002, 1,240,046 people were diagnosed with cancer in the United States (information from Centres for Disease Control and Prevention, 2004 and 2005, and National Cancer Institute, 2005). By way of example, according to Cancer Research UK, almost 44,100 cases of breast cancer are diagnosed in the UK each year (i.e. 16% of all new cancer cases), and over 12,400 deaths result annually from this disease in the UK. In the same period almost 7,000 new cases of pancreatic cancer are diagnosed in the UK (3% of all new cases), and approximately the same number of deaths result. An appreciation of why and how epigenetic changes are regulated is critical to the understanding, detection and treatment of cancer.
Methylation and resultant gene silencing plays an important role in cancer development and also in cancer progression. Reversal of the aberrant methylation patterns induced in cancer cells represents a way in which types of cancers that are poorly responsive to conventional chemotherapeutic treatments could be targeted. In addition, epigenetic treatment factors that target DNA methylation could also be used to treat advanced or inoperable cancers, or as a adjunct to other conventional existing therapies.
Somatic cell nuclear transfer (SCNT) is used to generate animals for livestock production (for cloning or for stem cell therapy), biomanufacturing of proteins and for disease modelling (Wilmut et al, 2002). A major obstacle to the application of SCNT in order to reprogramme somatic donor nuclei to a pluripotent state is the inefficient demethylation of the donor genome by the recipient oocyte. It has been found that genomic patterns of DNA methylation are reprogrammed genome-wide in early embryos and in primordial germ cells. The ability to manipulate in a targeted fashion epigenetic reprogramming in vivo may thus have important applications in regenerative medicine and in cancer therapy.
Several possible biochemical pathways for demethylation have been suggested, but until recently none of these has been proved to operate in vivo. In vitro studies in E. coli have shown that the cytidine deaminases Activation induced cytidine deaminase (Aid) and Apobec1 can deaminate 5-methylcytosine in DNA (Morgan et al 2004). This deamination results in a thymine base opposite a guanine, which may be repaired to a cytosine via endogenous DNA mismatch repair mechanisms, leading to effective demethylation. In the event that mismatch repair does not occur the deamination can cause transition mutations.
The role of Aid in antibody gene diversification and somatic hypermutation (SMH), via deamination of cytosines in specific regions of the immunoglobulin locus, has been previously characterised (Neuberger et al. 2003). In the organism, Aid is usually located in the cytoplasm where it is tightly regulated (Rada et al. 2002). It is believed that Aid activity is moderated by interactions with other proteins in the cell. Thus, it is generally thought that, in vivo, Aid is tightly controlled because an ‘unregulated’ deamination activity would be potentially hazardous to the cell since it could result in an increased rate of mutation in the genome and/or activation of epigenetically silenced genes.
It is apparent, therefore, that there is a need for novel compositions and methods that provide for either global or directed demethylation of genomic DNA in animal cells, optionally mammalian somatic cells, whilst maintaining the integrity of the sequence of that DNA.